recombinant chymosin
TRANSCRIPT
Lorenzo L. Taping IIIBS Biology
Disruption of PMR1 in Kluyveromyces lactisimproves secretion of
calf prochymosin
Chymosin
• Also known as rennin
• Main coagulating enzyme found in rennet which is used extensively in cheese production.
• hydrolyses a specific bond (Phe105–Met106) in kappa-casein of milk
• kappa-casein acts as micelle stabilizer
• Precipitation of insoluble constituents
Chymosin
Cheese curd
Whey
Chymosin
• Calf abomasums as the traditional principal source
Abomasum
Omasum
Reticulum
Rumen
Search for Alternative Rennet
• increasing demand • shortage of calf stomachs • ethical issues with animal
slaughtering
Kluyveromyces lactis
Increase chymosin production has been made through expression of calf chymosin gene in recombinant K. lactis.
Advantages of K. lactis
• Nontoxicogenic GRAS microorganism approved by US FDA
• Unlike P. pastoris, it does not require methanol to induce protein secretion
• Unlike E. coli, it doesn’t secrete the expressed protein enclosed in inclusion bodies
Further Improvements
Disruption of PMR1 in other yeast species (S. cerevisiae, P. pastoris, and H. polymorpha) increase the secretion of heterologous protein leading to the hypothesis that disrupting PMR1 in K. lactis would have the same effect.
PMR1• encodes for a P-type Ca2+-ATPase
localized in the Golgi apparatus• provides the yeast secretory
pathway with Ca2+ and Mn2+ required for glycosylation, sorting and endoplasmic reticulum-associated degradation of tagged proteins
• Improper sorting of synthesized proteins
• secretion of partially folded proteins• increase in the secretion of
heterologous proteins normally destined for degradation in the lysosome.
Defective PMR1
Experimentation
Microbial Strains K. lactis GG799 (type strain) E. coli JM109
Media Yeast Potato Dextrose for yeast Luria-Bertani for bacterium
Construction of K. lactis GG799 (pmr1∆)
Construction of expression vector
pKLAC1
BlnI SalI
pUC18-prochymosin
SalI BlnI
ligation
pKLAC1-N-prochymosin
Transformed into E. coli JM109
Prochymosin gene
Transformation of K. lactis GG799
SacII SacIILinearised cassette
Linearised cassette
K. lactis GG799
Transformed K. lactis GG799
transformants were selected using Yeast Carbon Base agar containing acetamide
Protein expression and chymosin activity assays
• Incubated in shake flasks containing YPD
• culture supernatants was added to 5mL of a suspension of non-fat milk powder and incubated until a clot formed
• One unit of chymosin activity was defined as the amount of active chymosin required to produce a clot in 40 min